Date of Award:

5-2015

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Chemistry and Biochemistry

Committee Chair(s)

Sean J. Johnson

Committee

Sean J. Johnson

Committee

Lance C. Seefeldt

Committee

Joan M. Hevel

Committee

Gregory J. Podgorski

Committee

Alvan C. Hengge

Abstract

A thorough understanding of protein function requires knowledge of how proteins interact with their substrates and with other proteins. The work entailed in this dissertation describes the binding interactions of proteins from two different model systems: (1) the dehydrogenase enzymes R- and S-HPCDH and (2) the zinc knuckle proteins Air1 and Air2.

R- and S-HPCDH are highly similar enzymes (42% identical) that function in a unique metabolic pathway found in the soil bacterium Xanthobacter autotrophicus. The bacterium produces R- and S-HPCDH simultaneously to facilitate the transformation of two different forms of the organic molecule epoxypropane to a common product that can be further metabolized and used as a source of energy for the microbe. R- and S-HPCDH are highly specific for either the right-handed or left-handed form (R- or S- forms) of their substrate molecules, respectively. Presented here are x-ray crystal structures (structural models) of S-HPCDH. Comparisons to the previously reported structure of R-HPCDH reveal structural differences that provide each enzyme with a distinct preference for binding and processing either the R- or S- form of their substrate molecules, demonstrating a structural basis for substrate preference by R- and S-HPCDH.

Air1 and Air2 are highly similar (45% identical) eukaryotic proteins that individually function within an essential three-protein complex called TRAMP. In the nucleus, TRAMP functions in RNA surveillance which is used to monitor different types of RNA molecules found in the nucleus and stimulate the degradation of any RNAs that need to be further processed or eliminated. Previous studies have indicated that Air1 and Air2 are involved in mediating crucial protein-protein interactions that link together the individual protein subunits of TRAMP. The work in this dissertation characterizes a previously unknown binding interface between Air2 and another TRAMP protein-component, the helicase Mtr4. Importantly, this interaction may explain how the functional activity of Mtr4 is modulated upon formation of TRAMP, a critical TRAMP functionality. In addition to protein interactions within TRAMP, this work has also identified a small region of Air1 that binds and regulates the activity of a protein that is not a part of TRAMP, the methyltransferase Hmt1. Collectively, these studies reveal important and previously unknown binding interactions of the multifaceted proteins Air 1 and Air2, and provide a foundation for future research efforts aimed at understanding their functions.

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